Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-gamma gene.

Th1 and Th2 CD4+ T cell clones have been defined by their ability to produce different lymphokines. However, the processes by which CD4+ T cells differentially regulate lymphokine gene expression have not been well defined. In this report, we demonstrate that the methylation status of a CpG dinucleotide contained within a TATA proximal regulatory element of the IFN-gamma promoter correlates with the transcription of the gene. In murine Th1 clones and two human CD4+ Th0 clones, this site is either completely or partially hypomethylated, whereas in murine Th2 clones this site is > 98% methylated. Treatment of murine Th2 clones with 5-azacytidine, an agent that inhibits methylation of the DNA, converts these cells to IFN-gamma producers. Additional targets for methylation outside the transcriptional control regions of the IFN-gamma genetic locus were found to be hypomethylated in Th2 cells but not in Th1 cells. Electrophoretic mobility shift assays (EMSA) revealed at least five distinct protein-DNA complexes that are formed with an oligonucleotide containing the IFN-gamma promoter TATA proximal regulatory element, and in vitro methylation of this site results in a loss of these three complexes. Furthermore, a comparison of nuclear extracts prepared from Th1 and Th2 clones revealed that the EMSA patterns were qualitatively similar but differed quantitatively. In addition, transient transfection of a murine IFN-gamma promoter-chloramphenicol acetyl transferase (CAT) gene construct into both Th1 and Th2 clones produced CAT activity that was not inducible by anti-CD3, indicating that hypomethylation per se of the promoter alone is not sufficient for inducible gene expression.